ABSTRACT HIV-1-infected T cells can form stable conjugates with non-infected T cells in a process known as ?virological synapse? formation. This process is reminiscent of the formation of an ?immunological synapse?, during which CD4+ T cells rapidly polarize the actin cytoskeleton, the microtubule-organizing center (MTOC), and cytokine- containing vesicles towards antigen-presenting cells. The polarization of CD4+ T cells during immunological synapse formation depends on the RHO family GTPase CDC42, a molecular switch that has a key role in the establishment of polarity in eukaryotic cells. We have now observed that CDC42 is critical for the efficient spreading of HIV-1 in several T cell lines and in primary cells. However, our data also imply that CDC42 is dispensable for the completion of a single cycle of replication. Together, our observations implicate CDC42 in the cell-to-cell transmission of HIV-1. CDC42 stimulates the formation of membrane extensions, such as filopodia, through effectors that mediate the polarization of the actin cytoskeleton, and HIV-1 can exploit filopodial bridges to spread from cell to cell. Thus, our data let us to propose a working model in which CDC42 is crucial for the formation of intercellular extensions that facilitate the transfer of HIV-1 between CD4+ T cells. In support of this model, we have observed that CDC42 is required for the formation of HIV-1-induced membrane extensions by MOLT-3 cells. An alternative working model is that CDC42 is required for the polarized trafficking of HIV-1 virion components to the virologic synapse. We propose to directly examine the roles of CDC42 in HIV-1 cell-to-cell transmission and virological synapse formation, and to determine whether HIV-1 regulates the activity of CDC42. We also propose to examine the roles of CDC42 effectors that regulate localized actin assembly and polarized trafficking in HIV-1 spreading. Among these effectors are F-BAR proteins that connect to actin polymerization machinery, as does the F-BAR protein PACSIN2, which we have recently implicated in the cell-to-cell transmission of HIV-1. Notably, our preliminary results indicate that certain CDC42 effectors, including the CDC42-regulated actin polymerase FMNL1 and a putative CDC42 effector that controls polarized exocytosis, have crucial roles in HIV-1 replication. The proposed studies have the potential to yield fundamental new insights into the mechanism of an important but poorly understood mode of HIV-1 transmission. Of particular significance would be the identification of a kinase downstream of CDC42 as being critical for HIV-1 spreading, since protein kinases constitute one of the most important groups of drug targets.